Collagenase type 4

The neuronal isolation and injection procedures have been previously described [9 (link)]. Briefly, SCG were dissected from adult Wistar rats and incubated in Earle’s balanced salt solution (Life Technologies, Rochelle, MD) with 0.55 mg/ml trypsin (Worthington, Freehold, NJ), 1.6 mg/ml Type IV collagenase (Worthington) for 1 hour at 35°C. Cells were then spun twice, transferred to minimum essential medium (Fisher Scientific, Pittsburgh, PA), plated, and incubated at 37°C until cDNA injection. cDNA injections was performed with an Eppendorf 5247 microinjector and Injectman NI2 micromanipulator (Madison, WI) 4–6 hours following cell isolation. Plasmids were stored at −20°C as a 1–2 μg/μl stock solution in TE buffer (10 mM TRIS, 1 mM EDTA, pH 8). The mGluR7, 8, and 4 clones (in pCDNA3.1+) were obtained from cDNA.org (Missouri S&T cDNA Resource Center, Rolla, MO). Concentrations of cDNAs injected were as indicated in the text. All neurons were co-injected with green fluorescent protein cDNA (0.02 μg/μl; pEGFPC1; Clontech Laboratories, Palo Alto, CA, USA) for identification of expressing cells. Cells were the incubated overnight at 37°C and experiments are performed the following day. All animal protocols were approved by the University of Rochester’s Committee on Animal Resources (UCAR).

Tumor specimens from 80 patients with PDAC were obtained from the Department of Surgery, SEOUL NATIONAL UNIVERSITY HOSPITAL (SNUH) and processed according to the guidelines provided by the ethics committee of SNUH (1503-093-657). Pathological features of all tissues were assessed according to WHO classification and AJCC staging (7th edition). The surgically dissected pancreatic tumor masses from patients with PDAC were washed with Phosphate-buffered saline (PBS) and minced into 3–5 mm² slices, and collected in RPMI 1640 supplemented with 10% FBS, 100 U/ml penicillin, and 100 U/ml streptomycin. Sliced tumor fragments were dissociated with 750 U/ml of type IV collagenase (Worthington Biochemical Corporation, Lakewood, NJ), and incubated for 1 h at 37°C in 5% CO₂, with rotation. After washing twice with RPMI 1640, a part of the cells was used for CD45 positive selection using magnetic-activated cell sorting, and the rest was used for culturing primary pancreatic cancer cells.

Single-cell suspensions of pulmonary cells were prepared as previously described (46 (link), 47 (link)). In brief, lung tissue was minced in 5 ml of 1× PBS containing 3 mg/ml type IV collagenase (Worthington). Samples were incubated at 37°C for 45 min and washed three times with 1× PBS. After digestion, residual red blood cells (RBCs) were removed with RBC lysis buffer. The total number of lung cells collected from each sample was determined by counting the cells in five squares of a counting chamber with an inverted light microscope at ×40 magnification. Lung cell suspensions were used for RNA extraction and flow cytometry (as described below), as well as for CFU determination by plating serial dilutions.

Brains of adult mice were minced and disaggregated in Type IV collagenase (400 U/ml, Worthington), Dispase (1.2 U/ml, Worthington), and DNase I (32 U/ml, Worthington) in PBS with Ca²⁺ and Mg²⁺ (GIBCO) at 37°C for 60 min, with gentle trituration every 10 min. After that, the disaggregated cells were washed with cold PBS and filtered through a 40‐μm mesh. After centrifugation, cell pellets were resuspended in 10 ml 20% BSA and centrifuged at 1000 g at 4°C for 25 min to remove the myelin. The pellets were then resuspended in PBS/3% FBS/0.1% NaN3/2 mM EDTA and blocked rat anti‐mouse CD16/32 (Mouse BD Fc Block, BD Pharmingen) for 5 min on ice and labeled with antibodies according to our previous study¹⁰ for 1 h at 4°C. The stained cells were sorted into EGM‐2/MV medium (Clontech) with an Aria II sorter (BD) or analyzed with an LSRII analyzer (BD) at Fluorescence‐activated cell sorting (FACS) Facility, and FACS data were analyzed using FlowJo software (TreeStar).

The cell isolation procedure from liver tissues was performed by magnetic based assay with some modifications 19. In brief, tissue explants were washed with phosphate buffer saline (PBS) and chopped into small pieces. After digestion with 0.05% type IV collagenase (Worthington Biochemical Corporation, NJ, USA, http://www.worthington-biochem.com) for 30 minutes at 37°C, tissues were again minced and the cell suspension was passed through a 70‐µm cell strainer (BD Biosciences, NJ, USA, https://www.bdbiosciences.com) to remove tissue debris. Red blood cells (RBCs) were lysed by 1x RBC lysis buffer (Stem Cell Technologies, Vancouver, Canada, https://www.stemcell.com) and the cells were washed again with PBS. An aliquot of the single cell suspension was used to determine the percentage of EpCAM+ cells by flow cytometry. Viable and nonviable cells were counted by trypan blue staining and then used to isolate EpCAM+ cells by magnet assisted cell sorting. In brief, depending on the cell number, they were labeled with appropriate amount of EpCAM+ selection cocktail using EpCAM+ Cell Isolation Kit (Stem Cell Technologies, Vancouver, Canada), according to manufacturer's instructions. Purity of EpCAM+ and EpCAM‐sorted cells were evaluated by flow cytometry.

Thirty C57BL/6 mice were injected intrasplenically with 1M Hepa1-6 cells and sacrificed at regular intervals to monitor for the development of liver tumors. In one mouse sacrificed 70 days after splenic injection, a liver tumor was observed. This tumor was excised and gently sliced and incubated at 37°C in DMEM containing type IV collagenase [0,35mg/mL] (Worthington Biochemical Corp. Lakewood, NJ, USA) and CaCl₂ [1mg/mL] for 15min under agitation. The cellular solution was then washed 3-times. The final cell suspension was seeded at a 0.25M cells/mL on COL1-coated dishes in supplemented medium. Medium was changed until cells reached confluence. Cells were then pooled and cultured in 75cm² flasks.